Immobilization of N-carbamyl-D-amino acid amidohydrolase

• Published in: Bioscience, biotechnology, and biochemistry Vol. 62; no. 10; pp. 1839 - 1844
• Main Authors: Nanba, H. (Kaneko Seed Co. Ltd., Maebashi (Japan)), Ikenaka, Y, Yamada, Y, Yajima, K, Takano, M, Ohkubo, K, Hiraishi, Y, Yamada, K, Takahashi, S
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 01.10.1998; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: ACIDE AMINE; AGROBACTERIUM; Amidohydrolases - biosynthesis; Amidohydrolases - chemistry; AMINO ACIDS; Amino Acids - chemical synthesis; AMINOACIDOS; Biological and medical sciences; Biotechnology; ENZIMAS INMOVILIZADAS; ENZYME IMMOBILISEE; Enzyme Stability; Enzymes, Immobilized - chemistry; ESCHERICHIA COLI; Escherichia coli - enzymology; Fundamental and applied biological sciences. Psychology; GENE; GENES; Hydrogen-Ion Concentration; IMMOBILISATION; IMMOBILIZATION; Immobilization of enzymes and other molecules; Immobilization techniques; IMMOBILIZED ENZYMES; INMOVILIZACION; LIASAS; LYASE; LYASES; Methods. Procedures. Technologies; N-carbamyl-D-amino acid amidohydrolase; Pseudomonas - enzymology; RESISTANCE A LA TEMPERATURE; RESISTENCIA A LA TEMPERATURA; Rhizobium - enzymology; TEMPERATURE RESISTANCE; thermotolerant; Stability; Support; Enzyme; Immobilization; Reducing agent; N-Carbamoyl-D-amino acid hydrolase; D-aminoacid; Agrobacterium; Enzymatic activity; Production; Bacteria; Hydrolases; Rhizobiaceae; Recombinant protein; Thermotolerance; Application; Immobilized enzyme; Comparative study
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.62.1839

N-Carbamyl-D-amino acid amidohydrolase (DCase, produced with recombinant Escherichia colt cells using a cloned gene from Agrobacterium sp. Strain KNK712, has been immobilized for use in the production of D-amino acids. The porous polymers, Duolite A-568 and Chitopearl 3003, were much better than other resins for the activity and stability of the adsorbed enzyme. The activity of DCase expressed on Duolite A-568 and Chitopearl 3003 amounted to 96 units/g-wet-resin and 91 units/g-wet-resin, respectively. DCase immobilized on Duolite A-568 was found to be most stable at about pH 7, and it was further stabilized by reductants such as dithiothreitol, L-cysteine, cysteamine, and sodium hydrosulfite. The stability during the repeated hatch reactions was greatly improved when dithiothreitol was in the reaction mixture, and the higher crosslinking degree with glutaraldehyde also stabilized the immobilized enzyme. After 14 times repeated reactions, the remaining activity of the immobilized enzyme cross-linked with 0.1% and 0.2% of glutaraldehyde, and 0.2% of glutaraldehyde with dithiothreitol in the reaction mixture was 12%, 18%, and 63%, respectively. DCase produced with Pseudomonas sp. strain KNK003A and Pseudomonas sp. strain KNK505, which are thermotolerant soil bacteria, and that with Agrobacterium sp. strain KNK712 were also immobilized on Duolite A-568. The stability of the enzymes of thermotolerant bacteria during reactions was superior to that of Agrobacterium sp. strain KNK712, though the activity was 1lower than that of strain KNK712